Excessive deposition of fats in hepatocytes causes inflammation and impairs insulin signaling, which in turn promotes the development of systemic insulin resistance and metabolic dysregulation. As the enzyme that phosphorylates adenosine, adenosine kinase (ADK) is expressed at the highest levels in the liver and critically determines the levels of both intracellular and extracellular adenosine. However, precisely how ADK regulates hepatic and systemic fat metabolism in relation to whole body insulin sensitivity is unknown. For this project, the ADK in hepatocytes has been validated to promote hepatic fat deposition and increase whole body fat mass in mice. Additional preliminary results also indicate: 1) hepatocyte-specific ADK overexpression in mice increases liver inflammation and causes systemic insulin resistance whereas hepatocyte-specific ADK disruption in mice decreases the severity of high-fat diet (HFD)-induced insulin resistance; 2) ADK disruption increases hepatic and macrophage expression of adenosine 2A receptor (A2AR), whose disruption increases hepatic ADK amount, exacerbates diet-induced hepatic fat accumulation and insulin resistance, impairs hepatocyte insulin signaling, and stimulates macrophage proinflammatory (M1) activation; and 3) DNA methylation was decreased in livers of hepatocyte-specific ADK-deficient mice and in macrophages from myeloid cell-specific ADK-deficient mice. Based on these findings, this project will test the central hypothesis that excessive ADK acts through dysregulating hepatocyte-macrophage crosstalk to promote hepatic fat deposition, impair liver insulin signaling, and increase liver inflammation, thereby bringing about hepatic and systemic insulin resistance. Mechanistically, ADK actions involve impaired A2AR signaling and increased DNA methylation within genes for hepatocyte fatty acid oxidation and macrophage anti-inflammatory responses. Accordingly, three Specific Aims will be pursued. Aim 1: Define the role of hepatocyte ADK in regulating fat metabolism and insulin sensitivity. In vivo experiments will be performed to examine the extent to which hepatocyte-specific ADK overexpression or disruption alters the severity of HFD-induced hepatic fat deposition, inflammation, and insulin resistance. In vitro experiments will be used to examine how ADK-driven hepatocyte factors alter the inflammatory status of macrophages/Kupffer cells. Aim 2: Define the role of macrophage ADK in regulating fat metabolism and insulin sensitivity. In vivo xperiments will be performed to examine the extent to which myeloid cell-specific ADK overexpression or disruption alters the severity of HFD-induced hepatic fat deposition, inflammation, and insulin resistance. In vitro experiments will be performed to examine whether and how ADK-driven macrophage factors promote hepatocyte fat deposition and inflammatory responses. Aim 3: Determine the extent to which ADK acts through decreasing A2AR signaling and/or increasing DNA methylation to impair hepatocyte fat metabolism and insulin signaling and enhance macrophage M1 activation. The successful completion of this project will accelerate the development of novel ADK inhibition-based approaches for managing obesity-associated chronic diseases.